Fam26c polymorphisms and use thereof for the diagnostic and treatment of late onset alzheimer&#39;s disease

ABSTRACT

Provided are methods of determining the likelihood that a subject will be diagnosed with Alzheimer&#39;s disease. Also provided are isolated and purified mammalian CALHM I, CALHM2, and CALHM3 proteins, vectors comprising a nucleic acid sequence encoding the CALHM 1, CALHM2, and CALHM3 proteins, and mammalian cells transfected with the vectors. Additionally, methods of affecting Ca2+ levels in a mammalian cell are provided. Further provided are methods of screening a test compound for the ability to alter calcium homeostasis in mammalian cells. Also, methods of affecting Ca2+levels in mammalian cells are provided. Additionally provided are methods of screening a test compound for the ability to inhibit ERK I/2 phosphorylation in a mammalian cell. Further provided are methods of screening a test compound for the ability to inhibit amyloid-beta peptide accumulation in a mammalian cell or biological fluid. Also provided are methods of screening for a test compound that may affect Alzheimer&#39;s disease.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention generally relates to genes and proteins that affect diseases in mammals. More specifically, the invention is directed to methods for determining the likelihood that a subject will develop Alzheimer's disease. The invention is also directed to proteins that affect Ca²⁺ transport in cells.

(2) Description of the Related Art

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a massive brain loss and by the presence of senile plaques and neurofibrillary tangles, two characteristic cerebral lesions formed by the aggregation of Aβ and tau proteins, respectively (Mattson, 2004; Selkoe, 2001). Sequential proteolysis of the amyloid-beta precursor protein (APP) by beta- and gamma-secretases produces two major Aβ species, Aβ40 and Aβ42. and increased Aβ production could represent a key determinant in amyloid formation and thus in the pathogenesis of AD.

The first atrophy observed in the AD brain occurs in the medial temporal lobe, which includes the hippocampus, and is the result of a massive synaptic degeneration and neuronal death (Braak and Braak, 1991). This early neurodegenerative process in the hippocampus is believed to lead to the characteristic learning and memory impairments observed in AD patients (de Leon et al., 2004). The etiology of the disease is complex because of its strong genetic heterogeneity (Marambaud and Robakis, 2005). Rare autosomal dominant mutations in the genes coding for the amyloid precursor protein (APP) and presenilins cause early-onset AD, whereas complex interactions between different genetic variants are believed to modulate the risk for the vast majority of late onset AD (LOAD) cases (Kennedy et al., 2003; Pastor and Goate, 2004). Concordant evidence of linkage to LOAD has been observed in different chromosomal regions, including on chromosome 10 where strong susceptibility loci are present (Kehoe et al., 1999; Bertram et al., 2000; Myers et al., 2000; Ertekin-Taner et al., 2000; Blaker et al., 2003; Farrer et al., 2003). Although significant associations with several candidate genes have been reported within these regions, the only susceptibility gene unambiguously demonstrated worldwide is the ε4 allele of APOE on chromosome 19 (Strittmatter et al., 1993). However, epidemiological studies indicate that the inheritance of the APOE ε4 allele cannot explain the overall heritability of AD, implying that a significant proportion of LOAD cases is attributable to additional genetic risk factors (Pastor and Goate, 2004).

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that increased risk for Alzheimer's disease is exhibited in individuals having a particular allele of a single nucleotide polymorphism (SNP) present in the FAM26C gene, renamed CALHM1 herein. The inventors have also characterized the CALHM1 protein as a Ca²⁺ ion channel that affects Ca²⁺ homeostasis, as well as Aβ accumulation in APP-transfected cells.

The present invention is directed to methods of determining the likelihood that a subject will be diagnosed with Alzheimer's disease. The methods comprise determining the subject's genotype at SNP rs2986017, where rs2986017 is at position 401 of SEQ ID NO:43. In these methods, an A at both of the subject's SNP rs2986017 alleles indicates an increased likelihood of an Alzheimer's disease diagnosis over a genotype at SNP rs2986017 that comprises a G at both alleles.

The invention is also directed to an isolated and purified mammalian CALHM1 protein, wherein the CALHM1 protein has an amino acid sequence at least 90% identical to SEQ ID NO:17.

Additionally, the invention is directed to a vector comprising a nucleic acid sequence encoding the above CALHM1 protein.

The invention is further directed to a mammalian cell transfected with the above vector.

The invention is additionally directed to an isolated and purified mammalian CALHM2 protein, where the CALHM2 protein has an amino acid sequence at least 90% identical to SEQ ID NO:16.

Also, the invention is directed to a vector comprising a nucleic acid sequence encoding the above CALHM2 protein.

The invention is further directed to a mammalian cell transfected with the above CALHM2 vector.

Additionally, the invention is directed to an isolated and purified mammalian CALHM3 protein, wherein the CALHM3 protein has an amino acid sequence at least 90% identical to SEQ ID NO:15.

The invention is further directed to a vector comprising a nucleic acid sequence encoding the above CALHM3 protein.

Also, the invention is directed to a mammalian cell transfected with the above CALHM3 vector.

The invention is additionally directed to methods of affecting Ca²⁺ levels in a mammalian cell. The methods comprise transfecting the cell with the above-described vector encoding a CALHM1.

The invention is further directed to other methods of affecting Ca²⁺ levels in a mammalian cell. The methods comprise transfecting the cell with the above vector encoding a CALHM2.

The invention is also directed to additional methods of affecting Ca²⁺ levels in a mammalian cell. The methods comprise transfecting the cell with the above vector encoding a CALHM3.

Also, the invention is directed to methods of screening a test compound for the ability to alter calcium homeostasis in a mammalian cell expressing a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. The methods comprise determining whether the test compound affects expression or activity of the CALHM1 protein. In these methods, a test compound that affects expression or activity of the CALHM1 protein has the ability to alter calcium homeostasis in the mammalian cell.

Additionally, the invention is directed to methods of screening a test compound for the ability to alter calcium homeostasis in a mammalian cell expressing a CALHM2 protein having an amino acid sequence at least 90% identical to SEQ ID NO:16. The methods comprise determining whether the test compound affects expression or activity of the CALHM2 protein. In these methods, a test compound that affects expression or activity of the CALHM2 protein has the ability to alter calcium homeostasis in the mammalian cell.

Further, the invention is directed to methods of screening a test compound for the ability to alter calcium homeostasis in a mammalian cell expressing a CALHM3 protein having an amino acid sequence at least 90% identical to SEQ ID NO:15. The methods comprise determining whether the test compound affects expression or activity of the CALHM3 protein. In these methods, a test compound that affects expression or activity of the CALHM3 protein has the ability to alter calcium homeostasis in the mammalian cell.

The invention is also directed to methods of affecting Ca²⁺ levels in a mammalian cell expressing a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. The methods comprising contacting the cell with a compound that affects expression or activity of the CALHM1 protein.

The invention is further directed to methods of affecting Ca²⁺ levels in a mammalian cell expressing a CALHM2 protein having an amino acid sequence at least 90% identical to SEQ ID NO:16. The methods comprising contacting the cell with a compound that affects expression or activity of the CALHM2 protein.

The invention is additionally directed to methods of affecting Ca²⁺ levels in a mammalian cell expressing a CALHM3 protein having an amino acid sequence at least 90% identical to SEQ ID NO:15. The methods comprising contacting the cell with a compound that affects expression or activity of the CALHM3 protein.

Also, the invention is directed to methods of screening a test compound for the ability to inhibit ERK1/2 phosphorylation in a mammalian cell. The methods comprise determining whether the test compound affects expression or activity of a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. In these methods, a test compound that affects expression or activity of the CALHM I protein has the ability to inhibit ERK1/2 phosphorylation in the mammalian cell.

Additionally, the invention is directed to methods of screening a test compound for the ability to inhibit amyloid-beta peptide accumulation in a mammalian cell or biological fluid. The methods comprise determining whether the test compound affects expression or activity of a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. In these methods, a test compound that affects expression or activity of the CALHM1 protein may have the ability to inhibit amyloid-beta peptide accumulation in the mammalian cell.

Further, the invention is directed to methods of screening for a test compound that may affect Alzheimer's disease. The methods comprise determining whether the compound affects expression or activity of a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. In these methods, a test compound that affects expression or activity of the CALHM1 protein may affect Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the alignment and phylogeny of CALHM1. Panel a shows a sequence alignment of human CALHM3 (SEQ ID NO:15), CALHM2 (SEQ ID NO:16), and CALHM1 (SEQ ID NO:17), and of murine and C. elegans CALHM1 (SEQ ID NO:18 and 19, respectively). Conserved sequences are shaded and sequence conservation is mapped in a shading gradient manner, the darkest shading representing the sequences of absolute identity and lighter shading representing the sequences of weaker conservation. Boxes denote hydrophobic domains 1-4 (HD1-4). * shows predicted N-glycosylation sites on human CALHM1. Panel b shows a phylogenetic tree including human CALHM1 (hCALHM1).

FIG. 2 is photographs of gels and western blots (WBs) and fluorescent micrographs of cells showing tissue expression, subcellular localization, and N-glycosylation of human CALHM1. For Panel a, total RNA was used for RT-PCR analyses targeting CALHM1 and β-actin transcripts in multiple human tissues (the first 20 lanes) and brain regions (the remaining 7 lanes on the right). Panel b shows immunofluorescence staining of permeabilized or non-permeabilized HT-22 cells transfected with human Myc-tagged CALHM1 (Myc-CALHM1) using anti-Myc (green) and anti-calreticulin (red) antibodies. Panel c shows HEK293 (lanes 1-3) and HT-22 (lanes 4-6) cells transfected with Myc-CALHM1 after incubation in the absence (−) or presence (+) of tunicamycin (Tunica) or N-glycosidase F (PNGase F). Lysates were probed with anti-Myc (upper panels) and anti-actin antibodies.

FIG. 3 is graphs and photographs of WBs showing that CALHM1 controls Ca²⁺ homeostasis by a mechanism that does not promote SOCE or activation of InsP₃Rs and RyRs. Panel a shows cytoplasmic Ca²⁺ measurements using Fluo-4 loading and “Ca²⁺ add-back” assays in HT-22 cells transiently transfected with Myc-CALHM1 or control vector. Cells were first incubated in Ca²⁺-free buffer (0 CaCl₂) and then challenged with physiological extracellular Ca²⁺ concentrations (1.4 mM CaCl₂) to monitor the progressive restoration of basal [Ca²⁺]_(i). The traces show the mean relative fluorescence units (RFU)+/−S.D. of three independent experiments. Insert, WB of the corresponding cell lysates probed with anti-Myc antibody; Vec, vector; C, CALHM1. Panel b shows peak and steady-state [Ca²⁺]_(i) measurements as in Panel a expressed in ΔF/F₀; *, P<0.001 (Student's t test). Panels c and d show cytoplasmic Ca²⁺ measurements as in Panel a in cells pretreated with the RyR inhibitor dantrolene [DTL, 10 μM (C)], the InsP₃R inhibitor xestospongin C [XeC, 2 μM (c)], and the two SOCE blockers 2-APB (50 μM) and Gd³⁺ (5 μM) (d). Panel e is WBs from “Ca²⁺ add-back” assays in vector- or Myc-CALHM1-transfected HT-22 cells preincubated in the absence (Control) or presence of PD98059 (PD98, 20 μM), EGTA (2 mM), and BAPTA-AM (20 μM). Cells were exposed to CaCl₂ for 30 min. Cell lysates were probed with antibodies directed against phosphorylated ERK1/2 (pERK1/2), total ERK1/2 (ERK1/2), and Myc (lower panels). To prevent rapid dephosphorylation by protein phosphatases, experiments were carried out in the presence of forskolin (30 μM) (Makhinson et al., 1999).

FIG. 4 is a sequence alignment diagram, photographs of WBs and graphs, showing pore-forming properties of CALHM1. Panel a shows WBs of lysates from non-transfected (NT) and Myc-CALHM1-tranfected HEK293 cells in the absence (Control) or presence of β-mercaptoethanol (βME) using anti-Myc (two upper panels) and anti-actin antibodies. Panel b shows WBs of lysates from HEK293 cells transfected (+) or not (−) with V5-tagged CALHM1 (V5-CALHM1) or Myc-CALHM1, after immunoprecipitatation with anti-Myc antibody. Total lysates (Input, left panels) and immunoprecipitates (Anti-Myc IP, right panels) were analyzed by WB using antibodies against V5 (upper panels), Myc (middle panels), and actin. Panel c shows a partial sequence alignment of human NMDAR NR2 (NMDAR2) subunits A-D and CALHM1 from various species. Sequence conservation is highlighted in a shading gradient as described above for FIG. 1A. * denotes Q/R/N site. The sequences, from top to bottom, are SEQ ID NO:20-42, respectively. Panel d shows cytoplasmic Ca²⁺ measurements in HT-22 cells transfected with control vector and wild type (WT) or N72G mutated Myc-CALHM1. Cells were treated and results analyzed as in FIG. 3A (n=3 independent experiments). Insert, WB of the corresponding cell lysates with anti-Myc antibody. Panel e shows peak [Ca²⁺]_(i) measurements as in Panel d, expressed in ΔF/F₀; *, P<0.001 (Student's t test).

FIG. 5 is graphs and photographs of WBs showing the CALHM1 P86L polymorphism impairs [Ca²⁺]_(i) and ERK1/2 phosphorylation. Panel a shows the cytoplasmic Ca²⁺ measurements in HT-22 cells transfected with control vector and WT or P86L mutated Myc-CALHM1. Cells were treated and results analyzed as in FIG. 3A (n=3 independent experiments). Insert, WB of the corresponding cell lysates with anti-Myc antibody. Panel b shows peak [Ca²⁺]_(i) measurements as in Panel a, expressed in ΔF/F₀; *, P<0.001 (Student's t test). Panel c shows western blots of HT-22 cells transfected with vector and WT or P86L mutated Myc-CALHM I analyzed by “Ca²⁺ add-back” assays and exposed to CaCl₂ for 30 min. Cell lysates were probed with antibodies directed against pERK1/2, ERK1/2, and Myc (lowest blot).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that increased risk for Alzheimer's disease is exhibited in individuals having a particular allele of the single nucleotide polymorphism (SNP) rs2986017, which is present in the FAM26C gene, renamed CALHM1 herein (Example 1). The inventors have also characterized the CALHM1 protein, and related proteins CALHM2 and CALHM3 as Ca²⁺ ion channels that affect Ca²⁺ homeostasis (Example 1). Additionally, CALHM1 protein was found to affect Aβ accumulation in APP-transfected cells (Example 2). These discoveries allow prediction of the relative risk of developing Alzheimer's disease. The discoveries also enable the manipulation of Ca²⁺ levels in cells using the CALHM1, CALHM2 and CALHM3 proteins.

The present invention is thus directed to methods of determining the likelihood that a subject will be diagnosed with Alzheimer's disease. The methods comprise determining the subject's genotype at SNP rs2986017, where rs2986017 is at position 401 of SEQ ID NO:43. In these methods, an A at both of the subject's SNP rs2986017 alleles (i.e., homozygous AA genotype at rs2986017) indicates an increased likelihood of an Alzheimer's disease diagnosis over a genotype at SNP rs2986017 that comprises a G at both alleles (i.e., homozygous GG genotype at rs2986017).

As described in Example 1, increased risk for Alzheimer's was conferred to the TT genotype at SNP rs2986017. This corresponds to the opposite strand AA genotype as described in the Genbank SNP database, describing the contig DNA at rs2986017, and provided as SEQ ID NO:43.

Preferably in these methods, an A at both of the subject's SNP rs2986017 alleles (i.e., AA homozygote) indicates an increased likelihood of an Alzheimer's disease diagnosis over a genotype at SNP rs2986017 that comprises a G at one or both alleles (i.e., GA heterozygote or GG homozygote). More preferably, an A at one or both of the subject's SNP rs2986017 alleles (i.e., AG heterozygote or AA homozygote) indicates an increased likelihood of an Alzheimer's disease diagnosis over a genotype at SNP rs2986017 that comprises a G at both alleles (i.e., GG homozygote).

The patient's genotype at rs2986017 can be linked to other SNPs, such that the genotype of the two SNPs are in linkage disequilibrium (LD) to each other. When the two SNPs are in LD, the two SNPs do not assort independently as in Hardy-Weinberg equilibrium (Balding, 2006). Under LD, the two SNPs are linked such that the prediction of the genotype at one SNP can be more and more reliably determined as LD increases by determining the genotype at the linked SNP. Thus, the genotype at a selected SNP can be reliably determined by determining the genotype at a SNP that is at high LD with the selected SNP.

The most common measures of LD are D′ and r² (Balding, 2006). With both of these measures, LD increases as D′ and r² approach 1.0. Thus, in these methods, the genotype at the selected SNP can be determined by determining the genotype at a second SNP that is at a high level of LD with the selected SNP.

In these methods, the patient's genotype at rs2986017 can thus be determined by determining the genotype at a secondary single nucleotide polymorphism (SNP) in linkage disequilibrium to rs2986017. Here, the linkage disequilibrium measure D′ between rs2986017 and the secondary SNP is greater than about 0.70, preferably greater than about 0.80, and more preferably greater than about 0.90 or 0.95 or 0.99.

The patient's genotype at rs29860I7 can also be determined by determining the genotype at a secondary single nucleotide polymorphism (SNP) in linkage disequilibrium to rs2986017, where the linkage disequilibrium measure r² between rs2986017 and the secondary SNP is greater than about 0.50, preferably greater than about 0.80, and more preferably greater than about 0.90 or 0.95 or 0.99.

The invention is also directed to an isolated and purified mammalian CALHM1 protein, wherein the CALHM1 protein has an amino acid sequence at least 90% identical to SEQ ID NO:17. Preferably, the CALHM1 protein has an amino acid sequence at least 99% identical to SEQ ID NO:17. More preferably, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17. In some embodiments, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17 except for an L86P substitution.

The CALHM1 protein here can be from any mammalian species, including rats, mice, or humans. The protein can also comprise a mutation or mutations that alter the protein's amino acid sequence, provided the resulting protein still retains Ca²⁺ ion channel activity.

Additionally, the invention is directed a vector comprising a nucleic acid sequence encoding the above CALHM1 protein. As used herein, a “vector” is a vehicle for delivering genetic material to a cell. The invention is not narrowly limited to any particular type of vector, Any such vector now known or later discovered may be utilized here, including, but not limited to, a plasmid vector or a viral vector. The skilled artisan would be capable of selecting the preferred vector for any particular purpose without undue experimentation. Preferably, the vector expresses the CALHM1 protein when transfected into a mammalian cell.

The invention is further directed to a mammalian cell transfected with the above vector. The cell here can be from any mammalian species, including rats and mice. Preferably, the cell is a human cell. The cell can be in culture, or preferably, the cell is in a living mammal.

The mammalian cell of these embodiments can be from any tissue type, including cells that naturally express CALHM1 and cells that do not. Preferably, the cell is a nerve cell or a brain cell. A preferred brain cell is a hippocampal cell. Other preferred cells are a spinal cord cell, a cerebral cortex cell, a cerebellum cell, a temporal lobe cell, a frontal lobe cell, and an occipital pole cell.

The invention is additionally directed to an isolated and purified mammalian CALHM2 protein, where the CALHM2 protein has an amino acid sequence at least 90% identical to SEQ ID NO:16. Preferably, the CALHM2 protein has an amino acid sequence at least 99% identical to SEQ ID NO:16. More preferably, the CALHM2 protein has an amino acid sequence completely identical to SEQ ID NO:16.

Also, the invention is directed a vector comprising a nucleic acid sequence encoding the above CALHM2 protein. Preferably, the vector expresses the CALHM2 protein when transfected into a mammalian cell.

The invention is further directed to a mammalian cell transfected with the above CALHM2 vector. The cell here can be from any mammalian species, including rats and mice. Preferably, the cell is a human cell. The cell can be in culture, or preferably, the cell is in a living mammal.

The mammalian cell of these embodiments can be from any tissue type, including cells that naturally express CALHM2 and cells that do not. Preferably, the cell is a brain cell, a uterine cell or a heart cell.

Additionally, the invention is directed to an isolated and purified mammalian CALHM3 protein, wherein the CALHM3 protein has an amino acid sequence at least 90% identical to SEQ ID NO:15. Preferably, the CALHM3 protein has an amino acid sequence at least 99% identical to SEQ ID NO:15. More preferably, the CALHM23 protein has an amino acid sequence completely identical to SEQ ID NO:15.

Also, the invention is directed a vector comprising a nucleic acid sequence encoding the above CALHM3 protein. Preferably, the vector expresses the CALHM3 protein when transfected into a mammalian cell.

The invention is further directed to a mammalian cell transfected with the above CALHM3 vector. The cell here can be from any mammalian species, including rats and mice. Preferably, the cell is a human cell. The cell can be in culture, or preferably, the cell is in a living mammal.

The mammalian cell of these embodiments can be from any tissue type, including cells that naturally express CALHM3 and cells that do not. Preferably, the cell is a placental cell.

The invention is additionally directed to methods of affecting Ca²⁺ levels in a mammalian cell. The methods comprise transfecting the cell with the above-described vector encoding a CALHM1.

The cell here can be from any mammalian species, including rats and mice. Preferably, the cell is a human cell. The cell can be in culture, or preferably, the cell is in a living mammal. The mammalian cell of these embodiments can be from any tissue type, including cells that naturally express CALHM1 and cells that do not.

The invention is further directed to methods of affecting Ca²⁺ levels in a mammalian cell. The methods comprise transfecting the cell with the above vector encoding a CALHM2.

The cell here can be from any mammalian species, including rats and mice. Preferably, the cell is a human cell. The cell can be in culture, or preferably, the cell is in a living mammal. The mammalian cell of these embodiments can be from any tissue type, including cells that naturally express CALHM2 and cells that do not.

The invention is also directed to methods of affecting Ca²⁺ levels in a mammalian cell. The methods comprise transfecting the cell with the above vector encoding a CALHM3.

The cell here can be from any mammalian species, including rats and mice. Preferably, the cell is a human cell. The cell can be in culture, or preferably, the cell is in a living mammal. The mammalian cell of these embodiments can be from any tissue type, including cells that naturally express CALHM3 and cells that do not.

Also, the invention is directed to methods of screening a test compound for the ability to alter calcium homeostasis in a mammalian cell expressing a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. The methods comprise determining whether the test compound affects expression or activity of the CALHM1 protein. In these methods, a test compound that affects expression or activity of the CALHM1 protein has the ability to alter calcium homeostasis in the mammalian cell. Preferably, the CALHM1 protein has an amino acid sequence at least 99% identical to SEQ ID NO:17. More preferably, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17. In some embodiments, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17 except for an L86P substitution.

Expression or activity of the CALHM1 protein can be determined by any method known in the art. For example, expression can be determined by determining levels of CALHM1 mRNA in the cell (e.g., by RT-PCR) or by quantifying the CALHM1 protein (e.g., by ELISA or western blot). Activity of the CALHM1 protein can be determined, e.g., by the methods described in Example 1.

These methods are not limited to testing any particular type of test compound. In some aspects, the test compound is a nucleic acid. An example is an aptamer that specifically binds to the CALHM1 protein. Preferably, the nucleic acid is complementary to a portion of the gene encoding the CALHM1 protein, e.g., an RNAi molecule (i.e., an miRNA, or any other small double-stranded RNA, now known or later discovered, that is capable of specifically interfering with expression of the target gene), an antisense molecule or a ribozyme.

The test compound in these methods can alternatively be a polypeptide, for example a protein that specifically binds to the CALHM1, preferably activating ion channel function. A preferred polypeptide test compound for these methods comprises an antibody binding site (e.g., a monoclonal antibody).

The test compound for these methods can also be an organic molecule less than about 1000 mw.

Additionally, the invention is directed to methods of screening a test compound for the ability to alter calcium homeostasis in a mammalian cell expressing a CALHM2 protein having an amino acid sequence at least 90% identical to SEQ ID NO:16. The methods comprise determining whether the test compound affects expression or activity of the CALHM2 protein. In these methods, a test compound that affects expression or activity of the CALHM2 protein has the ability to alter calcium homeostasis in the mammalian cell. Preferably, the CALHM1 protein has an amino acid sequence at least 99% identical to SEQ ID NO:16. More preferably, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:16.

Further, the invention is directed to methods of screening a test compound for the ability to alter calcium homeostasis in a mammalian cell expressing a CALHM3 protein having an amino acid sequence at least 90% identical to SEQ ID NO:15. The methods comprise determining whether the test compound affects expression or activity of the CALHM3 protein. In these methods, a test compound that affects expression or activity of the CALHM3 protein has the ability to alter calcium homeostasis in the mammalian cell. Preferably, the CALHM1 protein has an amino acid sequence at least 99% identical to SEQ ID NO:15. More preferably, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:15.

The invention is also directed to methods of affecting Ca²⁺ levels in a mammalian cell expressing a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. The methods comprise contacting the cell with a compound that affects expression or activity of the CALHM1 protein. Preferably, the CALHM1 protein has an amino acid sequence at least 99% identical to SEQ ID NO:17. More preferably, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17. In some embodiments, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17 except for an L86P substitution.

These methods are not limited to the use of any particular type of compound that affects expression or activity of the CALHM1 protein. In some aspects, the compound affects expression of the CALHM1 protein. Preferred such compounds are complementary to a portion of the gene encoding the CALHM1 protein and include an antisense molecule, a ribozyme or an RNAi molecule. Another preferred compound that affects the expression of the CALHM I protein is the vector described above that expresses the CALHM1 protein when transfected into a mammalian cell. Such a vector would increase expression of the CALHM1 protein.

In other aspects of these methods, the compound affects activity of the CALHM1 protein. Preferred such compounds comprise an antibody binding site, e.g., a monoclonal antibody that specifically binds to the CALHM1 protein, preventing CA²⁺ ion transport. The compound can also be an aptamer, e.g., that also specifically binds to the CALHM1 protein, preventing CA²⁺ ion transport. The compound can alternatively be an organic molecule less than about 1000 mw. In some embodiments, the compound used was identified by the above-described method of screening a test compound for the ability to alter calcium homeostasis in a mammalian cell expressing a CALHM1 protein.

The invention is further directed to methods of affecting Ca²⁺ levels in a mammalian cell expressing a CALHM2 protein having an amino acid sequence at least 90% identical to SEQ ID NO:16. The methods comprise contacting the cell with a compound that affects expression or activity of the CALHM2 protein. Preferably, the CALHM2 protein has an amino acid sequence at least 99% identical to SEQ ID NO:16. More preferably, the CALHM2 protein has an amino acid sequence completely identical to SEQ ID NO:16.

The invention is additionally directed to methods of affecting Ca²⁺ levels in a mammalian cell expressing a CALHM3 protein having an amino acid sequence at least 90% identical to SEQ ID NO:15. The methods comprise contacting the cell with a compound that affects expression or activity of the CALHM3 protein. Preferably, the CALHM3 protein has an amino acid sequence at least 99% identical to SEQ ID NO: 15. More preferably, the CALHM3 protein has an amino acid sequence completely identical to SEQ ID NO:15.

Also, the invention is directed to methods of screening a test compound for the ability to inhibit ERK1/2 phosphorylation in a mammalian cell. The methods comprise determining whether the test compound affects expression or activity of a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. In these methods, a test compound that affects expression or activity of the CALHM1 protein has the ability to inhibit ERK1/2 phosphorylation in the mammalian cell. Preferably, the CALHM1 protein has an amino acid sequence at least 99% identical to SEQ ID NO:17. More preferably, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17. In some embodiments, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17 except for an L86P substitution.

These methods are not limited to testing any particular type of test compound. In some aspects, the test compound is a nucleic acid. An example is an aptamer that specifically binds to the CALHM1 protein. Preferably, the nucleic acid is complementary to a portion of the gene encoding the CALHM1 protein, e.g., an RNAi molecule, an antisense molecule or a ribozyme.

The test compound in these methods can alternatively be a polypeptide, for example a protein that specifically binds to the CALHM1, preferably activating ion channel function. A preferred polypeptide test compound for these methods comprises an antibody binding site (e.g., a monoclonal antibody). The test compound for these methods can also be an organic molecule less than about 1000 mw.

Additionally, the invention is directed to methods of screening a test compound for the ability to inhibit amyloid-beta peptide accumulation in a mammalian cell or biological fluid. The methods comprise determining whether the test compound affects expression or activity of a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. In these methods, a test compound that affects expression or activity of the CALHM1 protein may have the ability to inhibit amyloid-beta peptide accumulation in the mammalian cell. Preferably, the CALHM1 protein has an amino acid sequence at least 99% identical to SEQ ID NO:17. More preferably, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17. In some embodiments, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17 except for an L86P substitution.

These methods are not limited to testing any particular type of test compound. In some aspects, the test compound is a nucleic acid. An example is an aptamer that specifically hinds to the CALHM1 protein. Preferably, the nucleic acid is complementary to a portion of the gene encoding the CALHM1 protein, e.g., an RNAi molecule, an antisense molecule or a ribozyme.

The test compound in these methods can alternatively be a polypeptide, for example a protein that specifically binds to the CALHM1, preferably activating ion channel function. A preferred polypeptide test compound for these methods comprises an antibody binding site (e.g., a monoclonal antibody). The test compound for these methods can also be an organic molecule less than about 1000 mw.

Further, the invention is directed to methods of screening for a test compound that may affect Alzheimer's disease. The methods comprise determining whether the compound affects expression or activity of a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO:17. In these methods, a test compound that affects expression or activity of the CALHM1 protein may affect Alzheimer's disease. Preferably, the CALHM1 protein has an amino acid sequence at least 99% identical to SEQ ID NO:17. More preferably, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17. In some embodiments, the CALHM1 protein has an amino acid sequence completely identical to SEQ ID NO:17 except for an L86P substitution.

These methods are not limited to testing any particular type of test compound. In some aspects, the test compound is a nucleic acid. An example is an aptamer that specifically binds to the CALHM1 protein. Preferably, the nucleic acid is complementary to a portion of the gene encoding the CALHM1 protein, e.g., an RNAi molecule (e.g., an miRNA, or any other small double stranded RNA, now known or later discovered, that is capable of specifically interfering with expression of the target gene), an antisense molecule or a ribozyme.

The test compound in these methods can alternatively be a polypeptide, for example a protein that specifically binds to the CALHM1, preferably activating ion channel function. A preferred polypeptide test compound for these methods comprises an antibody binding site (e.g., a monoclonal antibody). The test compound for these methods can also be an organic molecule less than about 1000 mw.

Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.

EXAMPLE 1 A Variant in CALHM1 Influences Ca²⁺ Homeostasis and Alzheimer Disease Risk Example Summary

DbEST was mined with TissueInfo for screening genes preferentially expressed in the hippocampus and located in linkage regions for Alzheimer disease. Reported here is the identification and characterization of CALHM1 on chromosome 10 that encodes a novel integral membrane glycoprotein controlling cytosolic Ca²⁺ levels and ERK/1/2 activation. CALHM1 was found to form homomultimers and to share striking sequence similarities with the ion selectivity filter of NMDA receptor. The conserved and functionally critical N72 residue in CALHM1 that aligns with the Q/R/N site of MNDA receptor was further identified. The common polymorphism P86L in CALHM1 caused impairments in Ca²⁺ homeostasis and was significantly over-represented in Alzheimer disease subjects in a large French case-control population. These data provide strong evidence that CALHM1 encodes an essential pore component of a novel ion channel family and constitutes a susceptibility gene for Alzheimer disease.

Introduction

Some neurodegenerative disorders are caused by mutations in genes almost exclusively expressed in the central nervous system. For instance, mutations in the brain proteins tau and α-synuclein, lead to autosomal dominant forms of frontotemporal dementia (Dermaut et al., 2005) and Parkinson's disease (Lee and Trojanowski, 2006), respectively. In this context, it was hypothesized that susceptibility for LOAD may come from genes predominantly expressed in affected brain regions, such as the hippocampus. By using TissueInfo (Skrabanek and Campagne, 2001) and the Alzgene database (Bertram et al., 2007) to screen for genes predominantly expressed in the hippocampus and located in linkage regions for LOAD, CALHM1 was identified. CALHM is designated a gene of unknown function, located on chromosome 10 at 1.6 Mb of the LOAD marker D10S1671 (Bertram et al., 2000). CALHM1 together with its two homologs, CALHM2 and CALHM3, represent the CALHM gene family and are clustered in 10q24.33. This work describes studies that show that CALHM1 homomultimerizes, controls cytosolic Ca²⁺ homeostasis, and shares similarities with the predicted selectivity filter of N-methyl-D-aspartate receptor (NMDAR). Importantly, it was also determined that the non-synonymous single nucleotide polymorphism (SNP) rs2986017 in CALHM1, which results in the P86L substitution, causes robust impairments in the regulation of cytosolic Ca²⁺ levels and in ERK1/2 phosphorylation. Further investigation determined that the frequency of the functional P86L polymorphism is significantly increased in a large cohort of AD cases in the French population. Here, it is proposed that CALHM1 is a pore component of a novel ion channel family of the brain and that variants in its gene family may constitute risk factors for LOAD. These results not only provide important new insights into the pathophysiology of cerebral Ca²⁺ homeostasis but also represent the first genetic evidence for a channelopathy component in AD etiology.

Results

Gene discovery. The human genome was screened with TissueInfo to annotate human transcripts with tissue expression levels derived from the expressed sequence tag database (dbEST) (Skrabanek and Campagne, 2001; Campagne and Skrabanek, 2006). Out of 33,249 human transcripts, the TissueInfo screen identified 30 transcripts whose expression was restricted to the hippocampus. These transcripts matched one to four ESTs sequenced from the hippocampus. Among these genes, a gene of unknown function previously annotated as FAM26C (Schneeberger et al., 2005) mapped to the AD locus on chromosome 10q and matched two hippocampal ESTs. This gene is hereafter referred to as CALHM1 (calcium homeostasis modulator 1). CALHM1 encodes an open reading frame of 346 amino acids predicted to contain four hydrophobic domains (HDs; TMHMM prediction) (Sonnhammer et al., 1998) and two N-glycosylation motifs (NetNGlyc 1.0 prediction) (Gupta and Jung, 2007) (FIG. 1A). No significant amino acid sequence homology to other functionally characterized proteins was found. Sequence database searches, however, identified five human homologs of CALHM1 (collectively identified as the FAM26 gene family) (Schneeberger et al., 2005). Two homologs of human CALHM1 are located next to CALHM1 on chromosome 10 and are designated CALHM2 (26% protein sequence identity) and CALHM3 (39% identity) (Schneeberger et al., 2005). CALHM1 is conserved across at least 20 species including mouse and C. elegans (see FIGS. 1A and 1B).

CALHM1 characterization. Using RT-PCR, we analyzed human CALHM1 gene expression in 20 tissues and six brain regions. The expression of CALHM1 was highest in the total adult brain and in all brain regions tested (FIG. 2A). CALHM1 expression was noticeably lower in all other tissues including fetal brain. No expression was detected in liver, heart, kidney, placenta, skeletal muscle, and uterus (FIG. 2A). Immunofluorescence staining in transiently transfected cells revealed that CALHM1 localizes predominantly to the endoplasmic reticulum (ER) where it colocalizes with the ER marker calreticulin (FIG. 2B). Immunofluorescence staining in non-permeabilized conditions revealed, however, the presence of several cells immunoreactive for CALHM1, indicating that a small pool of the protein reaches the cell surface (FIG. 2B). These data further show that the C-terminus end of the CALHM1 is extracellutarly oriented and so accessible to the anti-Tag antibody (FIG. 2B). Western blotting analyses revealed the presence of two immunoreactive bands in CALHM1 -transfected cells (FIG. 2C, lanes 2 and 5). Because CALHM1 is predicted to be N-glycosylated, it was asked whether these bands might represent different N-glycosylated forms of the protein. It was found that treatment with tunicamycin, which blocks cotranslational N-glycosylation within the ER, completely inhibited the appearance of the band of higher molecular weight and resulted in the maintenance of the lower band corresponding, therefore, to the unmodified core-protein (FIG. 2C, lanes 1-3). In vitro treatments of CALHM1 -transfected cell lysates with N-glycosidase F, which cleaves all types of asparagine bound N-glycans, also resulted in a molecular weight switch characteristic of protein deglycosylation (FIG. 2C, lanes 4-6). Thus, CALHM1 is a multipass transmembrane glycoprotein predominantly expressed in the adult brain and localized to the ER and plasma membranes.

CALHM1 controls cytosolic Ca²⁺ levels and ERK1/2 phosphorylation. TMHMM predicts that HD3 in CALHM1 is a re-entrant hydrophobic loop that does not cross the membrane bilayer, whereas HD1, HD2, and HD4 are membrane-spanning segments (Sonnhammer et al., 1998). In the absence of significant homology to other characterized proteins, it was postulated from the predicted topology that CALHM1 could represent an ion channel component. A suggestive similarity was indeed observed with the topology of ionotropic glutamate receptors, which also contain three transmembrane segments and a re-entrant loop that forms the lining of the pore region of the ion channels (Wollmuth and Sobolevsky, 2004). Because ionotropic glutamate receptors are Ca²⁺-transport membrane proteins (Gouaux and Mackinnon, 2005), it was asked whether CALHM1 could control cytoplasmic Ca²⁺ levels. Using Fluo-4 measurements in mouse hippocampal HT-22 cells, it was determined that transient expression of CALHM1 resulted in a robust and sustained increase in intracellular Ca²⁺ concentration ([Ca²⁺]_(i)) under extracellular “Ca²⁺ add-back” conditions (FIG. 3A). CALHM1 expression significantly increased the initial rate of change in [Ca²⁺]_(i) by forming a peak of fluorescence at ˜2 min following extracellular Ca²⁺ addition (FIGS. 3A and 3B, Peak). Expression of CALHM1 also induced a significant elevation in the steady-state [Ca²⁺]_(i), as compared to control conditions (FIG. 3B, Steady-state).

Cytosolic Ca²⁺ originates from the extracellular space or from intracellular stores, such as the ER (Berridge et al., 2003). Ca²⁺ release from the ER is mediated by ion channels, such as the inositol 1,4,5-triphosphate receptors (InsP₃Rs) or the ryanodine receptors (RyRs) (Id), whereas plasma membrane ion channels control Ca²⁺ influx. One important pathway of Ca²⁺ entry is coupled to ER Ca²⁺ release and is mediated by the mechanism of store operated Ca²⁺ entry (SOCE) (Lewis, 2007). We found that InsP₃R, RyR, or SOCE inhibitors had no effect on the [Ca²⁺]_(i) increase by CALHM1 (FIGS. 3C and 3D), indicating that CALHM1 does not promote Ca²⁺ influx or ER Ca²⁺ release by facilitating SOCE or activating InsP₃Rs and RyRs.

To address the physiological relevance of this observation, it was then asked whether CALHM1 expression could promote a Ca²⁺-dependent signaling pathway. Cytosolic Ca²⁺ is a remarkably versatile signal that controls multiple kinase-mediated pathways (Id.). ERK1/2 (extracellular signal-regulated kinases-1 and -2) became the focus because those kinases are involved in synaptic signaling in the adult brain and in the formation of long-term memories (Thomas and Huganir, 2004). Mechanistically, ERK1/2 are activated by phosphorylation by MEK1/2 (mitogen-activated protein kinase kinases-1 and -2) upon NMDAR-mediated Ca²⁺ influx during synaptic stimulation (Id). It was found that CALHM1 transient expression induced a robust increase in phosphorylated ERK1/2 (pERK1/2) levels under “Ca²⁺ add-back” conditions (FIG. 3E). This CALHM1-dependent increase in pERK1/2 was blocked by a MEK1/2 inhibitor and by intracellular Ca²⁺ chelation (FIG. 3E), showing that the stimulatory effect of CALHM1 on pERK1/2 is mediated by MEK1/2 and is dependent on Ca²⁺. Thus, CALHM1 promotes ERK1/2 phosphorylation by increasing [Ca²⁺]_(i) and activating MEK1/2.

CALHM1 has ion channel properties. Because many channels multimerize to form the ion pore (Ashcroft, 2006), and because monomeric CALHM1 cannot create a functional pore with three transmembrane segments, it was asked whether CALHM1 could form multimers. Western blot analysis of CALHM1-transfected cells under non-reducing conditions revealed the presence of immunoreactive bands with high molecular weights compatible with dimers and tetramers of CALHM1 (FIG. 4A). To test the possibility that CALHM1 self-associates, two differently tagged versions of the protein were co-expressed and co-immunoprecipitation experiments were undertaken to determine whether the two versions of CALHM1 form a complex. It was found that immunoprecipitation of a Myc-tagged CALHM1 co-precipitated with a V5-tagged CALHM1 (FIG. 4B), indicating that CALHM1 indeed homomultimerizes to form dimeric and possibly tetrameric structures.

Ionotropic glutamate receptors are ion-transport membrane proteins that operate in a selectively manner (Gouaux and Mackinnon, 2005). Recent advances made in the structural analysis of some ion channels have determined that ion selectivity is controlled by a short amino acid sequence called selectivity filter, which forms a narrow constriction in the pore across the membrane bilayer (Gouaux and Mackinnon, 2005; Doyle et al., 1998). The predicted selectivity filter of ionotropic glutamate receptors is located in the re-entrant loop called M2 and is critical for Ca²⁺ permeability (Wollmuth and Sobolevsky, 2004; Dingledine et al., 1999). By manual inspection, ionotropic glutamate receptor subunit sequences were screened for similarities with CALHM1. A short sequence was found in the C-terminus of CALHM1 HD2 that aligns with the predicted ion selectivity filter of NMDAR NR2 subunits (FIG. 4C). Previous studies have determined that the asparagine (N) residue in the so-called Q/R/N site of NMDAR NR2 subunits is critical for ion selectivity and permeation (see FIG. 4C, *) (Wollmuth and Sobolevsky, 2004). By sequence comparison, the highly conserved N72 residue was identified in human CALHM1 that aligns with the Q/R/N site at the C-terminus end of the second hydrophobic domain of both CALHM1 and NMDAR (FIG. 4C, *). Importantly, it was found that mutagenesis of the N72 residue to glycine (N72G) resulted in a significant inhibition of the effect of CALHM1 on [Ca²⁺]_(i) (FIG. 4D, E). Hence, CALHM1 shares striking similarities with the selectivity filter of NMDAR and the N72 residue is a key determinant in the control of cytosolic Ca²⁺ levels by CALHM1. Together with the observation that the effect of CALHM1 does not implicate known Ca²⁺ channels, these results strongly support the notion that CALHM1 is a novel pore-forming ion channel.

The CALHM1 P86L polymorphism is associated with LOAD. Because CALHM1 maps to a susceptibility region for LOAD, it was next tested whether CALHM1 SNPs are associated with LOAD. Two non-synonymous SNPs were already reported in databases, rs2986017 (+394 C/T; P86L) and rs17853566 (+927 C/A; H264N). CALHM1 exons were first sequenced in genomic DNA of 37 individuals, including 24 autopsy-confirmed AD cases and 13 age-matched normal controls. The rs17853566 SNP was not observed in this group. However, the presence of rs2986017 was confirmed (genotype distribution: CC=49%; CT=38%; TT=13%), with a potential over-representation of the TT genotype in AD subjects (AD=16.7%; Control=7.7%). In order to confirm this observation obtained in a very small sample, the impact of the rs2986017 SNP on the risk of developing LOAD was next assessed in a large French case-control population (710 LOAD cases and 565 controls, Table 1). The SNP distribution was in Hardy-Weinberg equilibrium in the control population (χ²=2.3; Table 1) but not in LOAD cases (χ²=15.1; Table 1). Importantly, the T allele distribution was significantly increased in LOAD cases (26%) as compared to controls (20%; P=0.0002; odds ratio=1.4). In addition, the TT genotype was found at a significantly higher frequency in LOAD subjects (10%) as compared to controls (5%; P=0.002; odds ratio=2.2; Table 1). The CALHM1 rs2986017 SNP is therefore significantly associated with an increased risk for AD in the French population tested. Consistently, we noticed that the patients bearing the TT genotype had an earlier age at onset compared with the CT and CC carriers (66.8±8.5 versus 68.7±7.7; P=0.05). In order to gain insight into the relevance of the rs2986017 SNP for the disease, the effect of the corresponding P86L substitution on Ca²⁺ homeostasis was investigated. Importantly, it was observed that the P86L mutation caused a significant inhibition of the effect of CALHM1 both on [Ca²⁺]_(i) (FIGS. 5A and 5B) and on ERK1/2 phosphorylation (FIG. 5C).

TABLE 1 Allele and genotype distribution of the CALHM1 P86L polymorphism in AD case and control populations Allele distribution Genotype distribution (%)¹ (%)² n C T CC CT TT AD cases 710 1051 (0.74) 369 (0.26) 410 231 69 (0.58) (0.32) (0.10) Control 565  907 (0.80) 223 (0.20) 370 167 28 (0.65) (0.30) (0.05) ¹P = 0.0002; ²P = 0.001 OR (T allele versus C allele) = 1.4, 95% CI [1.2-1.7], P = 0.0002 OR (CT + TT versus CC) = 1.4, 95% CI [1.1-1.8], P = 0.007 adjusted on age, gender and APOE status OR (CT versus CC) = 1.3, 95% CI [1.0-1.7], P = 0.08 adjusted on age, gender and APOE status OR (TT versus CC) = 2.2, 95% CI [1.3-3.6], P = 0.002 adjusted on age, gender and APOE status

Discussion

By tissue-specific data mining to screen for genes predominantly expressed in the hippocampus and located in linkage regions for LOAD, CALHM1, on chromosome 10, was identified. CALHM1 was found to encode an integral membrane glycoprotein containing several key characteristics of a Ca²⁺ release channel. CALHM1 controls cytosolic Ca²⁺ levels, homomultimerizes, and shares strong sequence similarities with the predicted selectivity filter of NMDAR (FIGS. 3 and 4). Importantly, it was also demonstrated that CALHM1 contains a functionally important N residue at position 72 that aligns with the Q/R/N site of the NMDAR selectivity filter (FIG. 4). Thus, NMDAR and CALHM1 share important structural similarities at the sequence level in a region that was previously described to be a critical determinant for Ca²⁺ selectivity and permeation by glutamate receptor ion channels (Wollmuth and Sobolevsky, 2004). Furthermore, it was shown that CALHM1 localizes to the cell surface where its C-terminal end is extracellularly oriented, suggesting that CALHM1 function may be regulated by extracellular ligands.

In the present report compelling evidence was provided that the rs2986017 SNP in CALHM1, which results in the P86L substitution, is associated with both an increased risk for LOAD and a dysregulation of Ca²⁺ homeostasis (Table 1 and FIG. 5). Specifically, it was shown that the CALHM1 P86L polymorphism leads to reduced levels of cytosolic Ca²⁺ and activated ERK1/2. A large body of literature supports the notion that a deranged intracellular Ca²⁺ signaling is occurring in AD and may be involved in neurodegeneration (Khachaturian, 1989; LaFerla, 2002; Mattson et al., 2000). However, it remains uncertain whether Ca²⁺ signaling interacts with pathways that involve the formation of neurofibrillary tangles (Davies, 2000) and senile plaques (Hardy and Selkoe, 2002), two characteristic cerebral lesions formed by the deposition of hyperphosphorylated tau protein and amyloid-β (Aβ peptide), respectively. The present results provide strong genetic evidence supporting the Ca²⁺ hypothesis of AD (Khachaturian, 1989; LaFerla, 2002; Mattson et al., 2000).

It is well established that highly regulated Ca²⁺ signals in hippocampal neurons control synaptic plasticity and memory formation by activating specific kinases, including ERK1/2 (Rao and Finkbeiner, 2007; Blitzer, 2005; Bardo et al., 2006). Indeed, upon excitatory neurotransmission several glutamate receptors, including NMDAR, are activated to trigger synaptic changes and memory storage by gating Ca²⁺ trough the postsynaptic membrane to promote kinase activation, gene transcription, and protein synthesis (Vao and Finkbeiner, 2007). These results have shown that CALHM1 is predominantly expressed in the brain and therefore suggest that the CALHM1 P86L polymorphism may critically impair neuronal Ca²⁺ homeostasis and the resulting ERK1/2-dependent transcriptional control of neurotransmission, a mechanism that could lead over time to the synaptic degeneration and neuronal loss observed in AD.

The present data further demonstrate the utility of tissue-specific data mining for identifying novel genes potentially involved in LOAD. Beside CALHM1, the screen has identified two additional candidate genes located in linkage regions on chromosomes 2 and 19. Interestingly, these genes are involved in the signaling by TGF-β and IGF receptors, two pathways critical for the control of ERK1/2 activation. This suggests the intriguing possibility that ERK1/2 signaling deregulation could represent a common feature for the disease susceptibility. Beyond AD genetics, however, our bioinformatics methods may have important ramifications in other research areas. Indeed, genome-wide association studies on large population samples represent so far the only reliable approach for identifying modest susceptibility variants for common and complex diseases. It is shown here that comparing tissue-specific gene expression profiles with genetic linkage data may represent a promising alternative screening strategy for identifying candidate genes for other disorders that affect isolated tissues or organs, such as heart disease or cancer.

CALHM1 is a member of a three-gene family whose members differ by their tissue expression profiles. While CALHM1 is mostly expressed in the brain (FIG. 2A), CALHM2 is predicted to be widely expressed. The following expression profiles were predicted with TissueInfo:

Predicted expression profile of CALHM2. Expressed most abundantly in uterus. Expression was also detected in pancreas, dorsal root ganglion, ganglion, muscle, corpus callosum, leukocyte, kidney, liver, gland, pancreatic islets, prostate, fibroblast, colon, mammary gland, amygdala, lung, thalamus, stem cell, artery, spleen, hippocampus, alveolar macrophage, thymus, eye, gut, skin, optic nerve, adrenal gland, heart, hypothalamus, ovary, cartilage, medulla oblongata, brain, placenta, testis, cervix, oligodendrocyte, subthalamus, bone, breast, adipose, epithelium, head, astrocyte, T cell and central ns. Thus, CALHM2 expression was found in heart, an organ where Ca²⁺ homeostasis is critical to normal physiology (Schneeberger et al., 2005).

Predicted expression profile of CALHM3. Expressed most abundantly in placenta. Expression also detected in lymphocyte and cervix.

Methods

TissueInfo tissue expression profiles. Known and predicted transcripts were obtained from Ensembl (human build NCBI35). Ensembl transcripts were filtered for repetitive sequence regions with RepeatBeater (graciously provided by Dr. Coward) (Schneeberger et al., 2005). Similarity searches between human ESTs and human Ensembl transcripts were conducted with megablast (Zhang et al., 2000). ESTs that matched transcripts with less than 95% sequence identity or over less than 150 base pairs were rejected (timegablast parameters—error 0.05-required-length 150-assemble-hsps). The resulting matches were processed with tiquery to produce whole genome tissue expression profiles. The programs timegablast and tiquery are from the TissueInfo distribution (Skrabanek and Campagne, 2001; icb.med.cornell.edu/crt/tissueinfo/index.xml). Whole genome profiles were filtered with InsightfulMiner 7.0 (Insightful Corp.) to extract the subset of transcripts annotated by TissueInfo as ‘specific to hippocampus’.

LOAD locus screen. The 30 transcripts predicted to be specific to hippocampus by TissueInfo were annotated with their genomic location using EnsMart/Biomart (Kasprzyk et al., 2004) using data from Ensembl. Chromosome numbers and FISH band locations were used to identify those transcripts that matched a locus of susceptibility for Alzheimer's Disease, as documented in AlzGene (Bertram et al. 2007).

Phylogeny prediction. Orthologs of CALHM1 were obtained from complete genomes available from Ensembl build 36 (Kasprzyk et al., 2004). A multiple sequence alignment of human CALHM1, CALHM2, CALHM3 and CALHM1 orthologs was constructed with T-coffe v 4.45 (NOtredame et al., 2002) and manually inspected. Phylogenetic trees constructed with JalView indicated an erroneous mouse ortholog assignment. The most likely CALHM1 mouse ortholog was found to be RefSeq XP_(—)921421. This sequence was used to construct the phylogenetic tree shown in FIG. 1. The phylogenetic tree was created with Phylip (Felsenstein, 2005) and the tree rendered as an unrooted tree with Phylodendron (iubio.bio.indiana.edu/treeapp/treeprint-form.html).

Materials and antibodies. Tunicamycin, PNGase F, GdCl₃, and β-mercaptoethanol were obtained from Sigma. Xestospongin C, 2-APB, dantrolene, PD98059, and BAPTA-AM were from Calbiochem. Forskolin was from MP Biomedicals. Anti-Myc antibody (clone 9E10) was from Chemicon and anti-calreticulin antibody from ABR Affinity BioReagents. Anti-actin antibody was from BD Transduction Laboratories. Anti-ERK1/2 and anti-phospho-ERK1/2 antibodies were from Cell Signaling Technology.

RT-PCR. Total human RNA preparations (1 μg, Clontech) from several brain regions (total brain, hippocampus, cerebellum, cerebral cortex, temporal lobe, frontal lobe, occipital pole) and 20 human tissues (Human Total RNA Master Panel II) were subjected to RT reactions using M-MLV-RT and random hexamer primers (Invitrogen). Ten percent of the RT reactions was used for the following PCR assays using GoTaq Flexi DNA polymerase (Promega). β-Actin PCR was performed with 0.4 μM primer (BAC1004: CTC CTT AAT GTC ACG CAC GAT TTC [SEQ ID NO:1] and BAC1008: GCC AAC CGC GAG AAG ATG ACC [SEQ ID NO:2]; Maxim Biotech) and 1.5 mM MgCl, under the following cycle conditions: 3 min denaturation at 94° C. and 30 cycles with 30 seconds at 94° C., 30 seconds at 58° C., 45 seconds at 72° C. Amplification of human CALHM1 was done with 0.4 μM primer F370 (5′-TGC TTC CTC TGT GCC TTC TG-3′-SEQ ID NO:3) and F777 (5′-CTC CAG GTC ATG GTT CAT GG-3′-SEQ ID NO:4) and 1.25 mM MgCl₂ under the following conditions: Denaturation for 3 min at 94° C. and 35 cycles with 30 seconds at 94° C., annealing for 30 seconds at 58° C., and extension for 30 seconds at 72° C. with a subsequent final extension at 72° C. for 10 min. PCR reactions were run in an Eppendorf Master gradient cycler.

CALHM1 subcloning and mutagenesis. Human CALHM1 cDNA (formerly annotated as FAM26C) was obtained from ATCC. The translated part of the cDNA was subcloned in frame with the carboxy-terminated Myc-His tag into pcDNA3.1 vector for overexpression experiments. To investigate protein oligomerization, CALHM1 was subcloned into pcDNA3.1-V5 tag vector. The P86L and N72G mutations were introduced by using the QuikChange II site-directed mutagenesis kit (Stratagene) and confirmed by sequencing of the entire CALHM1 insert.

Cell culture and transfections. All cell lines were tested negative for mycoplasma using MycoSensor PCR Assay Kit (Stratagene). Mouse hippocampal HT-22 cells were kindly provided by Dr. D. Schubert, Salk Institute, La Jolla, Calif. HEK293 cells were from ATCC. Cell lines were maintained in Dulbecco's Modified Earle's Medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine, and penicillin and streptomycin (Invitrogen). All cell lines were transiently transfected with wild type or mutated CALHM1 cDNAs at a cell density of about 50% using Lipofectamine PLUS reagent (Invitrogen) for HEK293 or Lipofectamine 2000 (Invitrogen) for HT-22 cells.

Immunofluoresence analysis. HT-22 cells grown on glass coverslips were transfected as described above. Cells were fixed five hours after transfection with 4% paraformaldehyde in Phosphate Buffered Saline (PBS) for 10 min at 37° C. Cells were then permeabilized or not with 0.1% Triton X-100 for 3 min at room temperature (RT) and blocked with Pierce Superblock in PBS. Cells were incubated at 37° C. with anti-Myc (1:100) and anti-calreticulin (1:2000) primary antibodies for 120 min, and with Alexa Fluor 488 and 594 anti-IgG secondary antibodies (1:2000, Molecular Probes) for 1 h. Cells were then visualized under a Nikon Eclipse TE2000-S fluorescent microscope.

Western blotting (WB) and immunoprecipitation (IP) assays. For WB, cells were washed with PBS and solubilized in ice-cold HEPES buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1X Complete protease inhibitor cocktail, Roche) containing 1% SDS. Ten micrograms of extracts was analyzed by SDS-PAGE. A standard ECL detection procedure was then used. For multimerization analyses and IP, cells were harvested six hours after transfection with the indicated CALHM1 cDNAs. Cells were then solubilized for 2 h at 4° C. in HEPES buffer containing 1% Nonidet P-40. Cell extracts were pre-cleared by centrifugation at 10,000 rpm for 5 min. For multimerization analyses, cell extracts were analyzed by WB in the absence (non-reducing conditions) or presence of 5% β-mercaptoethanol (reducing conditions). For IP, supernatants were immunoprecipitated with immobilized anti-Myc antibody, as per supplier's instructions (ProFound Mammalian c-Myc Tag IP Kit, Pierce). Total extracts and immunoprecipitated proteins were then analyzed by WB.

CALHM1 deglycosylation. HEK293 cells were transiently transfected for six hours with CALHM1 cDNA in the absence or presence of 10 μg/ml tunicamycin. CALHM1-transfected HT-22 cells were solubilized and incubated for 16 h at 37° C. in digestion buffer (50 mM NaH₂PO₄, pH 7.4, 20 mM EDTA, 0.2% SDS, and 1% β-mercaptoethanol) in the absence or presence of PNGase F. Cell lysates were then analyzed by WB using anti-Myc antibody, as described above.

Ca²⁺ measurements and “Ca²⁺ add-back” assays. Free cytosolic Ca²⁺ was measured in transiently transfected HT-22 cells plated in 6 well plates and loaded with the fluorescent Ca²⁺ indicator Fluo-4. 5.5 h post-transfection, cells were loaded with Fluo-4 as per manufacturer's recommendations (Fluo-4 NW Calcium Assay Kit, Molecular Probes). For “Ca²⁺ add-back” assays, cells were washed with Ca²⁺/Mg²⁺-free PBS and incubated for 10 min in the absence or presence of the indicated inhibitors in Ca²⁺/Mg²⁺-free Hanks' balanced salt solution (HBSS), supplemented with 20 mM HEPES buffer, 0.5 mM MgCl₂, and 0.4 mM MgSO₄. Ca²⁺ was then added back to a final concentration of 1.4 mM. Fluorescence measurements were obtained using a Tecan GENios Pro plate reader at 485 nm excitation and 535 nm emission. Experiments were carried out at RT. Cells were then washed with PBS and analyzed by WB.

CALHM1 sequencing. CALHM1 exons were completely sequenced using genomic DNA preparations obtained from 13 non-AD control individuals and 24 autopsy-confirmed AD patients. Subjects and genomic DNA preparations were described elsewhere (Conrad et al., 2002). Exons and intron/exon boundaries were amplified by PCR using the following primer sequences: FX1US 5′-TCT TGG AGG CAG CAG TGA GT-3′ (SEQ ID NO:5)(exon 1), FX1DSa 5′-TTT TGA GAG GTA GGG GGA TAG G-3′(SEQ ID NO:6)(exon 1) and FX2US 5′-GCT TTG GGA GTC TGA ACA GG-3′ (SEQ ID NO:7)(exon 2) FX2DS 5′-TCC TTT TTC CAC CTG GTT TG-3′ (SEQ ID NO:8)(exon 2). PCR conditions were as follows: Initial denaturation for 3 min at 94° C. and 35 cycles of 30 seconds at 94° C., annealing for 30 seconds at 54° C. (exon 1) or 53° C. (exon 2) and extension for 1 min at 72° C. for 35 cycles. ExoSAP purified PCR products were sequenced by GeneWiz.

SNP analyses—Population. The French AD and control subjects were all Caucasian (AD cases n=710, age at study=72.1±7.7 years, age at onset =68.7±8.I years, 38.7% male; Controls n=565, age=72.1±8.0 years, 39.4% male). A diagnosis of probable AD was established according to DSM-III-R and NINCDS-ADRDA criteria. Caucasian controls were defined as subjects without DMS-III-R dementia criteria, with integrity of cognitive function and with a MMS score≧25. Controls were recruited from retirement homes or from electoral rolls (altruistic volunteers). Each individual or next of kin gave informed consent. Control subjects with a family history of dementia were excluded.

Genotyping. The P86L genotype was determined by genomic DNA amplification of (i) a 114 by fragment using the forward mismatched primer 5′-GAAGAGTGGAAGCGGCCAC-3′ (SEQ ID NO:9) and reverse primer 5′-GACGGCCACCCAGACGACA-3′ (SEQ ID NO:10) following by Bsr I digestion and/or (ii) a 141 by fragment using the forward mismatched primer 5′-GAAGAGTGGAAGCGGCAGC-3′ (SEQ ID NO:11) and reverse primer 5′-GAGGAAGCATTTGCCGTCG-3′ (SEQ ID NO:12), followed by Alu I digestion. The genotyping of 176 individuals were checked by direct sequencing of a 207 by fragment using the forward primer 5′-CCTGGTGCTCTTTCTGCTTG-3′ (SEQ ID NO:13) and reverse primer 5′-CAGAAGGCAGAGGAAGCA-3′ (SEQ ID NO:14). Only two discrepancies were observed between CC and CT genotypes.

SNP analyses—Statistical analyses. The SAS software release 8.02 was used (SAS Institute, Cary, N.C.). Univariate analysis was performed using Pearson's χ² test. The allele and genotype distributions were considered different between the AD and control populations when p<0.05. The association of the P86L polymorphism with the risk of developing AD was assessed by a multiple logistic regression model adjusted for age, gender, and the APOE status. Interactions between age, gender, or APOE and the P86L polymorphism were tested by logistic regression. No significant statistical interactions were detected. Finally, the potential impact of the P86L polymorphism on age at onset was assessed using a general linear model.

Tissue Expression Profiles of CALHM2 and CALHM3. TissueInfo (Skrabanek and Campagne, 2001) was used to predict the expression of CALHM2 and CALHM3 in human tissues. The predicted expression profiles are respectively: CALHM2: Expressed most abundantly in uterus. Expression also detected in “pancreas, dorsal root ganglion, ganglion, muscle, corpus callosum, leukocyte, kidney, liver, gland, pancreatic islets, prostate, fibroblast, colon, mammary gland, amygdala, lung, thalamus, stem cell, artery, spleen, hippocampus, alveolar macrophage, thymus, eye, gut, skin, optic nerve, adrenal gland, heart, hypothalamus, ovary, cartilage, medulla oblongata, brain, placenta, testis, cervix, oligodendrocyte, subthalamus, bone, breast, adipose, epithelium, head, astrocyte, t cell, central ns”. CALHM3: Expressed most abundantly in placenta. Expression also detected in “lymphocyte, cervix”.

Notes. CALHM1 (also called FAM26C) has Ensembl accession code ENSG00000185933 (Uniprot Q81U99). CALHM3 (FAM26A; Ensembl ENSG00000183128; Uniprot Q86XJ0). CALHM2 (FAM26B; Ensembl ENSG00000138172; Uniprot Q9HA72). Genes with significant sequence similarity to CALHM1 in human include FAM26D (Uniprot Q5JW98), FAM26E (Uniprot Q8N5C1), and FAM26F (Uniprot Q5R3K3). Ensembl accession codes refer to Ensembl release 43.

Example 2 CALHM1 and Aβ Accumulation

In order to gain insight into the relevance of the rs2986017 SNP for the disease, the effect of the corresponding P86L substitution on Ca²⁺ homeostasis was investigated. Importantly, it was observed that the P86L mutation caused a significant inhibition of the effect of CALHM1 on [Ca²⁺]_(i) (FIGS. 5 a and 5 b). Cytosolic Ca²⁺ is a remarkably versatile signal that controls multiple pathways including APP metabolism (LaFerla, 2002). It was therefore asked whether CALHM1 P86L polymorphism affects Aβ levels in APP-transfected cells. While it was found that overexpression of wild type CALHM1 resulted in a robust decrease in the accumulation of both Aβ1-40 and Aβ1-42 under Ca²⁺ add-back conditions, P86L-mutated CALHM1 was unable to noticeably influence Aβ levels. These results demonstrate that CALHM1, by increasing cytosolic Ca²⁺, is able to repress Aβ accumulation. Strikingly, P86L polymorphism was found to lead to an inhibition of CALHM1 function resulting in a significant elevation of Aβ levels.

SEQ ID NOs DNA-artificial-β-actin PCR primer SEQ ID NO: 1 CTC CTT AAT GTC ACG CAC GAT TTC DNA-artificial-β-actin PCR primer SEQ ID NO: 2 GCC AAC CGC GAG AAG ATG ACC DNA-artificial-CALHM1 PCR primer SEQ ID NO: 3 TGC TTC CTC TGT GCC TTC TG DNA-artificial-CALHM1 PCR primer SEQ ID NO: 4 CTC CAG GTC ATG GTT CAT GG DNA-artificial-CALHM1 exon 1 PCR primer FX1US SEQ ID NO: 5 TCT TGG AGG CAG CAG TGA GT DNA-artificial-CALHM1 exon 1 PCR primer FX1DSa SEQ ID NO: 6 TTT TGA GAG GTA GGG GGA TAG G DNA-artificial-CALHM1 exon 2 PCR primer FX2US SEQ ID NO: 7 GCT TTG GGA GTC TGA ACA GG DNA-artificial-CALHM1 exon 2 PCR primer FX2DS SEQ ID NO: 8 TCC TTT TTC CAC CTG GTT TG DNA-artificial-PCR primer for P86L genotyping SEQ ID NO: 9 GAAGAGTGGAAGCGGCCAC DNA-artificial-PCR primer for P86L genotyping SEQ ID NO: 10 GACGGCCACCCAGACGACA DNA-artificial-PCR primer for P86L genotyping SEQ ID NO: 11 GAAGAGTGGAAGCGGCAGC DNA-artificial-PCR primer for P86L genotyping SEQ ID NO: 12 GAGGAAGCATTTGCCGTCG DNA-artificial-PCR primer for P86L genotyping SEQ ID NO: 13 CCTGGTGCTCTTTCTGCTTG DNA-artificial-PCR primer for P86L genotyping SEQ ID NO: 14 CAGAAGGCAGAGGAAGCA Protein-Human CALHM3/FAM26A SEQ ID NO: 15   1 mdkfrmlfqh fqsssesvmn giclllaavt vklyssfdfn cpclvhynal yglgllltpp  61 lalflcglla nrqsvvmvee wrrpaghrrk dpgiirymcs svlqralaap lvwillalld 121 gkcfvcafss svdpekfldf anmtpsqvql flakvpcked elvrdspark aysrylrcls 181 qaigwsvtll liiaaflarc lrpcfdqtvf lqrrywsnyv dleqklfdet ccehardfah 241 rcvlhffasm rselqarglr rgnagrrlel pavpeppavp eppegldsgs gkahlraiss 301 reqvdrllst wysskppldl aaspglcggg lshraptlal gtrlsqhtdv Protein-Human CALHM2/FAM26B SEQ ID NO: 16   1 maaliaenfr flslffkskd vmifnglval gtvgsqelfs vvafhcpcsp arnylyglaa  61 igvpalvlfi igiilnnhtw nlvaecqhrr tkncsaaptf lllssilgra avapvtwsvi 121 sllrgeayvc alsefvdpss ltareehfps ahateilarf pckenpdnls dfreevsrrl 181 ryesqlfgwl ligvvailvf ltkclkhycs plsyrqeayw aqyranedql fqrtaevhsr 241 vlaannvrrf fgfvalnkdd eelianfpve gtqprpqwna itgvylyren qglplysrlh 301 kwaqglagng aapdnvemal lps Protein-Human CALHM1/FAM26C SEQ ID NO: 17   1 mmdkfrmifq flqsnqesfm ngicgimala saqmysafdf ncpclpgyna aysagillap  61 plvlfllglv mnnnvsmlae ewkrplgrra kdpavlrymf csmaqralia pvvwvavtll 121 dgkcflcafc tavpvsalgn gslapglpap elarllarvp cpeiydgdwl larevavryl 181 rcisqalgws fvllttllaf vvrsvrpcft qaaflkskyw shyidierkl fdetctehak 241 afakvciqqf feamnhdlel ghthgtlata pasaaapttp dgaeeerekl rgitdqgtmn 301 rlltswhkck pplrlgqeep plmgngwagg gprpprkeva tyfskv Protein-Mouse CALHM1/FAM26C SEQ ID NO: 18   1 mdkfrmifqf lqsnqesfmn gicgimalas aqmysafdfn cpclpgynvv yslgilltpp  61 lvlfllglvm nnnismlaee wkrpagrrak dpavlrymfc smaqraliap vvwvavtlld 121 gkcflcafct avpvatlgng slvpglpape larllarvpc peiydgnwll arevavrylr 181 cisqalgwsf vllttllafv vrsvrpcftq vaflkskyws hyidierklf detctehaka 241 fakvciqqff eamnhdlelg hthgvlatat atatateavq spsdrteeer eklrgitdqg 301 tmnrlltswh kckpplrlgq eaplmsngwa ggeprpprke vatyfskv Protein-C. elegans CALHM1/FAM26C-GenBank NP_495403 SEQ ID NO: 19   1 mttsinsvvt vfqnvftnhg stllngilia ttvggqslvr kltfscpcay piniyhslvf  61 mfgptaalll igitvnsttw klahgfffrv rdtrhswktt cvswievliq ssvapiawlf 121 vvfldggyyr cyrshefcli sdailcknst ilnsyastss fnkisdngky cppcicvpnp 181 tdasyleaes qiyawglllf sgvaaflvit cnrmcdkytl vqrqyvetyk nvetqkfdav 241 akehasqlae hnaraffgqk dwtkrdwdwv sgipevnnpl farlrliaae ktqqtmytpl 301 qlwndnkgyr ipqpdlqltq iivdetked Protein-Human NMDAR2D (partial sequence) SEQ ID NO: 20 siwllwalvfnnsvpven Protein-Human NMDAR2C (partial sequence) SEQ ID NO: 21 svwllwalvfnnsvpien Protein-Human NMDAR2B (partial sequence) SEQ ID NO: 22 aiwllwglvfnnsvpvqn Protein-Human NMDAR2A (partial sequence) SEQ ID NO: 23 aiwllwglvfnnsvpvqn Protein-Human CALHM1 (partial sequence) SEQ ID NO: 24 lvlfllglvmnnnvsmla Protein-Chimpanzee CALHM1 (partial sequence) SEQ ID NO: 25 lvlfllglvmnnnvsmla Protein-macaque CALHM1 (partial sequence) SEQ ID NO: 26 lvlfllglvmnnnvsmla Protein-Opossum CALHM1 (partial sequence) SEQ ID NO: 27 lvlfllglvmnnnvsmla Protein-Elephant CALHM1 (partial sequence) SEQ ID NO: 28 lvlfllglvmnnnvsmla Protein-Cat CALHM1 (partial sequence) SEQ ID NO: 29 lvlfllglvmnnnvsmla Protein-Cow CALHM1 (partial sequence) SEQ ID NO: 30 lvlfllglvmnnnvsmla Protein-Rat CALHM1 (partial sequence) SEQ ID NO: 31 lvlfllglvmnnnismla Protein-Mouse CALHM1 (partial sequence) SEQ ID NO: 32 lvlfllglvmnnnismla Protein-Dog CALHM1 (partial sequence) SEQ ID NO: 33 lvlfllglvmnnnvsvla Protein-Platypus CALHM1 (partial sequence) SEQ ID NO: 34 avlfllglvmnnnvsmla Protein-Armadillo CALHM1 (partial sequence) SEQ ID NO: 35 lllfllglvlnnnvsmla Protein-Hedgehog CALHM1 (partial sequence) SEQ ID NO: 36 lllfllglvlnnnvsmla Protein-Chicken CALHM1 (partial sequence) SEQ ID NO: 37 lilfllgfvlnnnvsmla Protein-Zebrafish CALHM1 (partial sequence) SEQ ID NO: 38 iwffllgfvlnnnvsmla Protein-Medaka CALHM1 (partial sequence) SEQ ID NO: 39 iwffllgfvlnnnvsvla Protein-Tetraodon CALHM1 (partial sequence) SEQ ID NO: 40 iwffmlgfvlnnnvsvla Protein-Fugo CALHM1 (partial sequence) SEQ ID NO: 41 iwffmlgfvlnnnvsvla Protein-Stickleback CALHM1 (partial sequence) SEQ ID NO: 42 vwffligfvlnnkvsvlt DNA-Human-SNP rs2986017 SEQ ID NO: 43 ACCTGAGCAG AGGCCCCATT TTGAGAGGTA GGGGGATAGG GCCCTCCCAG AGGGACCTTG ATCTGCCAGG GAGACCCAGC GTGAAGCCAT GCGGCCCCTC ACCTGGGAGA TGCAGCGGAG GTAACGCACG GCCACCTCTC GGGCCAACAG CCAGTCGCCA TCGTAGATCT CAGGGCAGGG CACCCGGGCC AGCAGGCGGG CGAGCTCGGG GGCAGGAAGG CCGGGTGCCA GGCTGCCGTT GCCCAGTGCG CTCACGGGCA CGGCAGTGCA GAAGGCACAG AGGAAGCATT TGCCGTCGAG TAGCGTGACG GCCACCCAGA CGACAGGCGC GATGAGGGCG CGCTGGGCCA TGGAGCAGAA CATGTAGCGC AACACAGCGG GGTCCTTGGC CCGGCGGCCC (A/G) GCGGCCGCTT CCACTCTTCG GCCAGCATGG ACACGTTGTT GTTCATGACC AGGCCAAGCA GAAAGAGCAC CAGGGGTGGC GCCAGCAGGA TGCCCGCGCT GTAGGCTGCA TTGTAGCCCG GCAGGCAGGG GCAGTTGAAG TCGAAGGCCG AGTACATCTG GGCACTGGCC AGGGCCATGA TGCCACAGAT GCCATTCATG AAGGACTCCT GGTTGGACTG CAGGAACTGG AAGATCATCC GGAACTTGTC CATCATGCCC GCTGTGGGGC CCGGCCTCCT CTTCCCAACT CACTGCTGCC TCCAAGAGGG CCCCTGCTGC CCACCCTGCC CACTGGGTGC CCACCTCATG ACTCGGGCTC TCCTGGCTGG GACCAACAGA GCTCAGAGCA GAGGCTGAGG 

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In view of the above, it will be seen that the several advantages of the invention are achieved and other advantages attained.

As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

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1. A method of determining the likelihood that a subject will be diagnosed with Alzheimer's disease, the method comprising determining the subject's genotype at SNP rs2986017, wherein rs2986017 is at position 401 of SEQ ID NO:43, wherein an A at both of the subject's SNP rs2986017 alleles indicates an increased likelihood of an Alzheimer's disease diagnosis over a genotype at SNP rs2986017 that comprises a G at both alleles.
 2. The method of claim 1, wherein an A at both of the subject's SNP rs2986017 alleles indicates an increased likelihood of an Alzheimer's disease diagnosis over a genotype at SNP rs2986017 that comprises a G at one or both alleles.
 3. The method of claim 1, wherein an A at one or both of the subject's SNP rs2986017 alleles indicates an increased likelihood of an Alzheimer's disease diagnosis over a genotype at SNP rs2986017 that comprises a G at both alleles.
 4. The method of claim 1, wherein the patients genotype at SNP rs2986017 is determined by determining the genotype at a secondary SNP in linkage disequilibrium to SNP rs2986017, wherein the linkage disequilibrium measure r² between SNP rs2986017 and the secondary SNP is greater than 0.50.
 5. The method of claim 4, wherein the linkage disequilibrium measure r² between the SNP rs2986017 and the secondary SNP is greater than 0.80.
 6. The method of claim 4, wherein the linkage disequilibrium measure r² between the SNP rs2986017 and the secondary SNP is greater than 0.90.
 7. An isolated and purified mammalian CALHM protein, wherein the CALHM protein is (i) a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO: 17, (ii) a CALHM2 protein having an amino acid sequence at least 90% identical to SEQ ID NO: 16, or (iii) a CALHM3 protein having an amino acid sequence at least 90% identical to SEQ ID NO:
 15. 8-10. (canceled)
 11. A vector comprising a nucleic acid sequence encoding the CALHM protein of claim
 7. 12-40. (canceled)
 41. A method of affecting Ca 2″+⁺, levels in a mammalian cell, the method comprising transfecting the cell with the vector of claim
 11. 42-46. (canceled)
 47. A method of screening a test compound for the ability to alter calcium homeostasis in a mammalian cell expressing a CALHM protein the method comprising determining whether the test compound affects expression or activity of the CALHM protein, wherein a test compound that affects expression or activity of the CALHM protein has the ability to alter calcium homeostasis in the mammalian cell, wherein the CALHM protein is (i) a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO: 17, (ii) a CALHM2 protein having an amino acid sequence at least 90% identical to SEQ ID NO: 16, or (iii) a CALHM3 protein having an amino acid sequence at least 90% identical to SEQ ID NO:
 15. 48-61. (canceled)
 62. A method of affecting Ca²⁺ levels in a mammalian cell expressing a CALHM, the method comprising contacting the cell with a compound that affects expression or activity of the CALHM protein, wherein the CALHM protein is (i) a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO: 17, (ii) a CALHM2 protein having an amino acid sequence at least 90% identical to SEQ ID NO: 16, or (iii) a CALHM3 protein having an amino acid sequence at least 90% identical to SEQ ID NO:
 15. 63-79. (canceled)
 80. A method of screening a test compound for the ability to inhibit ERK 1/2 phosphorylation in a mammalian cell, the method comprising determining whether the test compound affects expression or activity of a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO: 17, wherein a test compound that affects expression or activity of the CALHM1 protein has the ability to inhibit ERK 1/2 phosphorylation in the mammalian cell. 81-88. (canceled)
 89. A method of screening a test compound for the ability to inhibit amyloid-beta peptide accumulation in a mammalian cell or biological fluid, the method comprising determining whether the test compound affects expression or activity of a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO: 17, wherein a test compound that affects expression or activity of the CALHM1 protein may have the ability to inhibit amyloid-beta peptide accumulation in the mammalian cell. 90-97. (canceled)
 98. A method of screening for a test compound that may affect Alzheimer's disease, the method comprising determining whether the compound affects expression or activity of a CALHM1 protein having an amino acid sequence at least 90% identical to SEQ ID NO: 17, wherein a test compound that affects expression or activity of the CALHM1 protein may affect Alzheimer's disease. 99-106. (canceled) 